This invention relates to gel electrophoresis and more particularly to methods and apparatuses for detecting bands in gels, such as for example gel scanners.
In one class of gel scanner, bands representing molecular species that have been resolved in the gel by electrophoresis are detected optically by determining the amount of certain frequencies of light that are absorbed by the band. The absorption of light is aided by staining the bands. This class of gel scanner includes at least one light source and a photodetector. Commonly, a filter is used to select the frequency of light absorbed by the bands. A reference signal is obtained from a photocell responding to light which is not subject to absorption by the bands that have been resolved within the gel before being transmitted to the photocell.
In one prior art gel scanner of this type, one light beam is transmitted through the gel to detect bands and a second beam of light is transmitted through gel in which there are no bands. The two beams of light are detected by photocells and the signals from them compared to determine when light has been absorbed by a band of molecular species being resolved in the gel.
This prior art gel scanner has a disadvantage in that its sensitivity or its ability to differentiate bands from gel is adversely affected by background such as by bubbles in the gel which background may be different in the area through which the sensing beam of light passes and the area through which the reference beam passes.
In another type of prior art photometric device, two different narrow bandwidth frequencies of light are used. One frequency is within the range that is absorbed by the sample and the other is not in that range. The two frequencies are applied together to the sample to make the measurement and then separated, with one of the frequencies being used as the sensing frequency and the other being used as a reference frequency to remove background noise.
This type of prior art photometric device has a disadvantage in that it is expensive, inconvenient to use, and under some circumstances provides a substantial amount of nonlinearity.
The deficiencies of prior art gel scanners have been so large that they have not generally been used to scan silver stained gels. Silver stained gels have been photographed instead. This is time consuming and furthermore destroys some of the high sensitivity which is the principal advantage of silver staining conventional stains. The difficulty in scanning silver stained gels arises from the fact that silver stains do not produce light absorbance bands of narrow wavelength or frequency bandwidth. In fact, they are brown in color.
Photography can cope with this since photographs can be made with the gel backilluminated with a physically broad, spatially uniform light source that lessens the photometric effect of gel imperfections, such as bubbles. On the other hand, direct scanning of the gel must necessarily use a narrow, collimated light source, such as through pairs of slits. Because of the broad wavelength bandwidth of absorbance of silver stains, the usual signal-to-noise improvement method of using a narrow sensing wavelength bandwidth won't work since it responds only to a small part of the total absorbance. On the otherhand, this bandwidth can't be sufficiently increased without prohibitive linearity problems. In the subject invention, this problem is solved by using a broad bandwidth of reference wavelengths.